|
| Status |
Public on Nov 04, 2015 |
| Title |
HepG2 exposed to APAP, rep1 |
| Sample type |
RNA |
| |
|
| Source name |
HepG2 exposed to APAP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
cell type: hepatic cell line
exposed to: acetaminophen
|
| Treatment protocol |
Hepatic in vitro models, i.e. HepaRG, HepG2 and hSKP-HPC were exposed to acetaminophen for 24 hours at concentrations (IC10) of 13mM, 2mM and 18mM, respectively. Corresponding vehicle controls were incubated to the same medium without acetaminophen.
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| Growth protocol |
hSKP-HPC: hSKP cells were cultivated using growth medium composed of DMEM+GLUTAMAX/F12 Nutrient Mixture (3:1; Life Technologies) supplemented with 7.33 IU/mL benzyl penicillin (Continental Pharma), 50 mg/mL streptomycin sulfate (Sigma-Aldrich), 2.5 mg/mL fungizone, 2 % (v/v) B27 Supplement (Life Technologies), 40 ng/mL basic fibroblast growth factor (FGF) 2 (Promega) and 20 ng/mL epidermal growth factor (EGF) (Promega). Approximately two weeks after isolation, hSKP spheroids were dissociated to single cells using Liberase DH (Roche). The cells were subsequently cultivated as a moolayer. Hepatic differentiation of hSKP was carried out using a 24-day protocol in which cells were exposed in a time-dependent manner to the following growth factors and cytokines: Activin A (Life Technologies), FGF4 (Biosource), bone morphogenetic protein 4 (BMP4), hepatocyte growth factor (HGF) (Life Technologies), insulin-transferin-sodium selenite solution (ITS) (Sigma-Aldrich), dexamethasone (Sigma-Aldrich) and oncostatin M (Life Technologies). The obtained differentiated hepatocyte-like cells are referred to as hepatic progenitor cells (hSKP-HPC). HepG2: HepG2 cells (ATCC; clone HB-8065) were recovered from liquid nitrogen and were cultured in 75 cm2 tissue culture flasks (Falcon) in a humidified incubator (37oC, 5% (v/v) CO2). The cell culture medium was composed of DMEM (Lonza) containing 10% (v/v) bovine calf serum (Gibco). The cells were passaged at sub confluence at least three times before use in exposure experiments. A seeding density of approximately 13,000 cells / cm2 was employed each time. HepaRG: cryo-preserved differentiated HepaRG cells were obtained from Biopredic International and cultivated as per the supplier's instructions. The cells were cultivated using basal hepatic medium supplemented with HepaRG Maintenance and Metabolism Supplement (Biopredic International). During the exposure experiments the basal hepatic medium was supplemented with HepaRG Induction Supplement (Biopredic International).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The isolation was conducted using the GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma-Aldrich) following the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
Total RNA (100 ng) was used for amplification and in vitro transcription using the Genechip 3’ IVT Express Kit following the manufacturer’s instructions (Affymetrix). The amplified RNA was purified with magnetic beads and 15 μg Biotin-aRNA was treated with fragmentation reagent.
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| |
|
| Hybridization protocol |
12.5 μg fragmented aRNA was hybridized to Affymetrix Human Genome U133 plus 2.0 arrays along with a hybridization cocktail solution and then placed in a Genechip Hybridization Oven-645 (Affymetrix) rotating at 60 rpm at 45 ºC for 16 h. After incubation, arrays were washed on a Genechip Fluidics Station-450 (Affymetrix) and stained with the Affymetrix HWS kit as indicated by the manufacturer’s protocols.
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| Scan protocol |
The chips were scanned with an Affymetrix Gene-Chip Scanner-3000-7G and the quality control matrices were confirmed with the Affymetrix GCOS software following the manufacturer’s guidelines.
|
| Description |
HepG2_APAP1
|
| Data processing |
Background correction, summarization (median polish) and normalization (quantile) were done with Robust Multi-array Analysis using RMAExpress v1.0.5
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| |
|
| Submission date |
Oct 14, 2015 |
| Last update date |
Nov 06, 2015 |
| Contact name |
Robim M Rodrigues |
| E-mail(s) |
rmarceli@vub.ac.be
|
| Organization name |
Vrije Universiteit Brussel
|
| Department |
IVTD
|
| Street address |
Laarbeeklaan 103
|
| City |
Brussels |
| ZIP/Postal code |
1090 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE74000 |
Gene expression data from acetaminophen-induced toxicity in human hepatic in vitro systems and clinical liver samples |
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